SPE-GC-MS for the sampling and determination of unmetabolized styrene in urine

The urinary excretion of unmetabolized styrene can be a very good indicator for biomonitoring styrene in occupationally exposed people. The use of a new urine sampling system, involving a solid-phase extraction cartridge, offers several advantages for determining styrene. The advantages are especially related to the pre-analytical phase of styrene determination, which may be influenced by many variables. The effect on styrene recovery of sorbent type, eluting solvent, elution volume, elution flow-rate, and the addition of methanol to the washing solvent, was evaluated by experimental design methodology. As a result, Oasis HLB cartridges were selected for urine sampling, as well as 1.5 mL of ethyl acetate at 0.5 mL/min for eluting the retained styrene. These conditions were then applied to the validation of the solid-phase extraction combined with GC-MS method for the sampling and analysis of unmetabolized styrene in urine. The overall uncertainty was in the 12-22% range and the limit of detection was 2.2 microg/L for a 4 mL urine sample. The stability of styrene has been studied both in cartridges and in vials under different storage periods. After 1 month period the styrene stored on cartridges at room temperature remained stable, whereas this is not the case for styrene recovery from vials. The results obtained indicate that on-site solid-phase extraction of urine can provide a simple, accurate and reproducible sampling and analytical method for the biomonitoring of styrene in urine.     

均匀设计法优化高丽参多糖的提取

利用均匀设计法优化了高丽参多糖的提取过程.主要考察料液质量比、浸泡时间、提取温度和提取时间四个因素对提取率的影响.通过数学模型得到最优提取状况和3D曲面图.结果表明,提取温度对高丽参多糖得率影响最大,在料液质量比1:35、浸泡时间135 min、水提取温度81℃条件下提取156 min,高丽参多糖最大理论提取率为49.51%.最优化模型下实际多糖得率49.29%与理论得率相符.     

HS-GC/MS法分析乌龙茶挥发性成分

本试验旨在采用静态顶空-气相色谱/质谱法(HS-GC/MS)建立起一种快速测定分析以金观音为代表的乌龙茶中挥发物的方法。本方法利用单因素试验结合L9(34)正交试验,以出峰个数和总峰面积为考察指标,确定了顶空平衡温度、平衡时间、茶/Na Cl以及加样量4种因素的最佳萃取条件,并以此方法对金观音的挥发性成分进行了分析。结果表明,平衡温度对出峰个数和总峰面积的影响最大,而平衡时间的影响最小。试验得出的顶空进样的最优条件为:平衡温度80℃,平衡时间60 min,茶/Na Cl为1∶2,加样量为0. 5 g。在此萃取条件下能够得到104种物质,这些物质能更真实地反映金观音香气成分的化学构成,为以金观音为代表的乌龙茶的开发利用提供有价值的数据。     

Multivariate optimisation of microwave-assisted extraction of capsaicin from Capsicum frutescens L. and quantitative analysis by 1H-NMR

A simple and rapid microwave-assisted extraction (MAE) procedure combined with 1H-NMR spectrometry was developed and optimised for the extraction and quantitative determination of capsaicin in Capsicum frutescens. The influence of experimental variables, including irradiation power, extraction temperature and dynamic extraction time before reaching the selected extraction temperature, on the performance of the extraction procedure was systematically studied using a Box-Behnken experimental design followed by a conventional central composite design approach. Statistical treatment of the results together with results from some additional experiments suggested optimum extraction conditions as 120 degrees C and 150 W, for 15 min with acetone as extractant. The optimised MAE method provides extracts that can be analysed quantitatively using 1H-NMR without any preliminary clean-up or derivatisation steps. In the 1H-NMR spectrum of the crude extracts the doublet signal in the delta range 4.349-4.360 ppm was well separated from other resonances in deuterated chloroform. The quantity of the compound was calculated from the relative ratio of the integral value of the target peak to that of a known amount of dimethylformamide as internal standard. In comparison with traditional Soxhlet extraction, the proposed method is less labour-intensive and provides a drastic reduction of extraction time and solvent consumption. In addition, MAE showed higher extraction yield and selectivity, with comparable reproducibility and recovery, relative to both conventional Soxhlet and sonication methods.     

Optimization of extraction of high-ester pectin from passion fruit peel (Passiflora edulis flavicarpa) with citric acid by using response surface methodology

A central composite design was employed to optimize the extraction of pectin with citric acid. The independent variables were citric acid concentration (0.086–2.91% w/v) and extraction time (17–102 min). The combined effect of these variables on the degree of esterification was investigated. Results have shown that the generated regression models adequately explained the data variation and significantly represented the actual relationship between the independent variables and the responses. Besides that, the citric acid concentration was the most important factor to affect the degree of esterification, as it exerted a significant influence on the dependent variable. Lower citric acid concentration increased the pectin degree of esterification. The surface response showed the relationships between the independent variables, and thus responses were generated. Through this surface, the satisfactory condition of 0.086% w/v citric acid for 60 min was established for extraction of high-ester yellow passion fruit pectin.     

Influence of extraction parameters on the mutagenicity of soil samples

The aim of this study was to investigate the influence of four extraction parameters (type of solvent, temperature, duration of extraction, and soil mass/solvent volume ratio) on the mutagenicity of soil extracts. Four urban soil samples were submitted to the micro-method adaptation of the Ames test on Salmonella typhimurium according to the following sequence: identification of the most sensitive strain (TA98 or TA100), the best solvent(s), the optimum extraction temperature and extraction time, and finally the optimal soil/solvent ratio. Extraction was thus performed using eight different solvents (distilled water, dichloromethane, acetonitrile, acetone, cyclohexane, methanol, hexane, or ethanol), two temperatures (room temperature or 37 degrees C), two durations (4 or 24 h), and two soil mass/solvent volume ratios (1:2 or 1:10). The results show that strain TA98 was more sensitive than strain TA100, and the observed mutagenicity was expressed as number of TA98 revertants per mg of soil equivalent. No mutagenicity was induced by the distilled water extracts, whereas most of the organic solvent extracts induced a significant mutagenic response. A dichloromethane/acetone mixture appeared to be the best compromise for extraction of mutagens from the urban soils tested. Moreover, the present study showed that a higher mutagenic activity was generally obtained with a temperature of 37 degrees C (compared to room temperature), with an extraction time of 24 h (compared to 4 h), and with a soil mass/solvent volume ratio of 1:10 (compared to 1:2).     

Ultrasound Assisted Extraction of Phenolic Compounds from Peaches and Pumpkins

The ultrasound-assisted extraction (UAE) method was used to optimize the extraction of phenolic compounds from pumpkins and peaches. The response surface methodology (RSM) was used to study the effects of three independent variables each with three treatments. They included extraction temperatures (30, 40 and 50°C), ultrasonic power levels (30, 50 and 70%) and extraction times (10, 20 and 30 min). The optimal conditions for extractions of total phenolics from pumpkins were inferred to be a temperature of 41.45°C, a power of 44.60% and a time of 25.67 min. However, an extraction temperature of 40.99°C, power of 56.01% and time of 25.71 min was optimal for recovery of free radical scavenging activity (measured by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) reduction). The optimal conditions for peach extracts were an extraction temperature of 41.53°C, power of 43.99% and time of 27.86 min for total phenolics. However, an extraction temperature of 41.60°C, power of 44.88% and time of 27.49 min was optimal for free radical scavenging activity (judged by from DPPH reduction). Further, the UAE processes were significantly better than solvent extractions without ultrasound. By electron microscopy it was concluded that ultrasonic processing caused damage in cells for all treated samples (pumpkin, peach). However, the FTIR spectra did not show any significant changes in chemical structures caused by either ultrasonic processing or solvent extraction.     

Monitoring protein expression by proteomics: human plasma exposed to benzene

Low levels and long term exposure to benzene is associated with hematotoxicity including aplastic anemia, acute myelogenous leukemia, and lymphoma. Current biomonitoring methods such as urinary phenol, S-phenylmercapturic acid, and trans-trans muconic acid were found to be unreliable as analytical methods to detect benzene exposure. Therefore, to search for a specific protein for biomonitoring benzene exposure, we investigated plasma proteins from workers (n = 50) at a printing company who were exposed to benzene, by two-dimensional gel electrophoresis. The protein profiles are significantly different (p < 0.05) between benzene exposed and unexposed groups, as identified by matrix-assisted laser desorption ionization/time of flight mass spectrometry and confirmed by Western blot analyses. T cell receptor beta chain (TCR beta), FK506-binding protein, and matrix metalloproteinase-13 were expressed only in benzene exposed workers. In addition, interleukin-4 receptor alpha chain and T cell surface glycoprotein CD1b precursor were found to be up-regulated in the plasma of benzene exposed workers. When we treated Jurkat cells with benzene (10 microM-10 mM), TCR beta expression was increased in the membrane more than 6-9-fold compared to untreated cells. In addition, the amount of TCR beta released into the culture media, at benzene concentrations greater than 50 microM, increased up to 10 mM. Therefore, TCR beta levels in plasma could be used as a biomarker and a possible therapeutic target for benzene exposure.     

Determination of valproic acid in human serum and pharmaceutical preparations by headspace liquid-phase microextraction gas chromatography-flame ionization detection without prior derivatization

An efficient and fast extraction technique for the enrichment of valproic acid from human blood serum samples using the headspace liquid phase microextraction (HS-LPME) combined with gas chromatography (GC) analysis has been developed. The extraction was conducted by suspending a 2 microL drop of organic solvent in a 1 mL serum sample; following 20 min of extraction, withdrawing organic solvent into a syringe and injection into a GC with a flame ionization detector (FID), without any further pre-treatment. Four organic solvents, 1-decanole, benzyl alcohol, 1-octanol and n-dodecane, were studied as extractants, and n-dodecane was found to be the most sensitive solvent for valproic acid. The results revealed that HS-LPME is suitable for the successful extraction of valproic acid from human blood serum samples. Parameters like extraction time, ionic strength, pH, organic solvent volume, and temperature of the sample were studied and optimized to obtain the best extraction results. An enrichment factor of 27-fold was achieved in 20 min. The procedure resulted in a relative standard deviation of <13.2% (n=7) and a linear calibration range from 2 to 20 microg mL(-1) (r>0.98), and the limit of detection was 0.8 microg mL(-1) in serum blank samples. Overall, LPME proved to be a fast, sensitive and simple tool for the preconcentration of valproic acid from real samples. The proposed method was also applied to the analysis of valproate in pharmaceutical preparations.     

黑曲霉发酵花生根提取白藜芦醇的优化

本研究利用黑曲霉发酵花生根产生的纤维素酶来酶解花生根中的纤维,破坏其细胞壁,有利于自藜芦醇的提取。同时对发酵后的花生根白藜芦醇的提取进行优化。根据中心组合实验设计原理,采用四因素五水平的响应面分析法优化发酵后花生根中自藜芦醇的提取,响应面分析表明乙醇浓度、提取温度、提取时间和液固比对花生根白藜芦醇提取率有显著影响,且不是简单的线性关系。得到最佳的提取条件为乙醇浓度64％,提取温度55℃,提取时间60min,液固比8：1,在此条件下,自藜芦醇得率为0．191％。     

超声提取-高效液相色谱法测定甘草中甘草酸含量

确定了制备甘草酸分析样品的超声波提取条件, 0.3%稀氨水为溶剂,液固比50:1,提取时间5 h;确定了高效液相色谱法检测甘草酸含量的条件为ODS色谱柱(4.6mm×25 cm, 5 μm),检测波长254 nm,检测温度为室温,流动相CH3OH/3% HOA c(V/V)为75/25,流速1mL/m in,进样量10 μL,平均加样回收率100.20%,相对标准偏差为1.77%。结果表明超声提取-高效液相色谱法是一种准确度高,速度快的测定甘草中甘草酸含量的检测方法。     

Determination of tramadol in human plasma and urine samples using liquid phase microextraction with back extraction combined with high performance liquid chromatography

Liquid phase microextraction by back extraction (LPME-BE) combined with high performance liquid chromatography (HPLC)-fluorescence detection was developed for the determination of tramadol in human plasma. Tramadol was extracted from 2 mL of basic sample solution (donor phase) with pH 11.5 through a micro liter-size organic solvent phase (100 microL n-octane) for 25 min and finally into a 3.5 microL acidic aqueous acceptor microdrop with pH 2.5 suspended in the organic phase from the tip of a HPLC microsyringe needle for 15 min with the stirring rate of 1250 rpm. After extraction for a period of time, the microdrop was taken back into the syringe and injected into HPLC. Effected the experimental parameters such as the nature of the extracting solvent and its volume, sample temperature, stirring rate, volume of the acceptor phase, pH and extraction time on LPME-BE efficiency was investigated. At the optimized condition, enrichment factor of 366 and detection limit (LOD) of 0.12 microg L(-1) were obtained. The calibration curve was linear (r=0.999) in the concentration range of 0.3-130 microg L(-1). Within-day relative standard deviation RSD (S/N=3) and between-day RSD were 3.16% and 6.29%, respectively. The method was successfully applied to determine the concentration of tramadol in the plasma and urine samples and satisfactory results were obtained.     

响应面法优化微波辅助提取山苍子核仁油的研究

通过微波辅助提取技术结合响应面法优化山苍子核仁油提取条件,以期建立更高产率的提取方法。该研究在单因素设计基础上,选取液料比、微波功率、萃取时间、萃取温度4个主要因素,分析这4个因素对山苍子核仁油提取率的影响。结果表明:通过建立多元回归拟合分析,得出山苍子核仁油提取最佳工艺条件为液料比1∶16,萃取温度为69℃,微波功率为337 W,萃取时间为63 min,在此条件下山苍子核仁油提取率为37.42%,与环己烷溶剂回流法相比较提取率提高了30.11%。气质联用仪分析结果显示,山苍子核仁油主要成分有16种占总成分的88.21%,鉴定出10种脂肪酸占总成分的78.24%,饱和脂肪酸有4种占总成分的43.23%,不饱和脂肪酸有6种占总成分的35.01%,脂肪酸中含量最高的为月桂酸(31.36%)。该研究结果表明该方法严谨、可靠,采用微波辅助提取山苍子核仁油是可行的。     

Evaluation of antitumour activity of tea carbohydrate polymers in hepatocellular carcinoma animals

Box-Behnken design criterion was applied to identify the significant effects of various extraction parameters such as temperature, time, and solvent-solid ratio on extraction of tea carbohydrate. Among the three variables tested extraction temperature, and solvent-solid ratio were found to have significant effect on tea carbohydrate extraction. The most suitable condition for extraction of tea carbohydrate was found to be a single step extraction at extraction temperature 90°C, extraction time 30 min, and solvent-solid ratio 5:1. At these optimum extraction parameters, the maximum yield of tea carbohydrate obtained experimentally was found to be very close to its predicted value of 3.47% dry weight of root. Then, we have studied the influence of tea carbohydrate on biochemical parameters in hepatocellular carcinoma (HCC) animals. Hepatocellular carcinoma was induced by the injection of 1×10(5) H22 hepatocarcinoma cells into right hind thigh muscle in experimental animals. Tea carbohydrate could inhibit tumour growth and decrease microvessel density in tumour tissue. The altered amount of serum white blood cells (WBC), Interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in HCC animals were dose-dependently increased, whereas activities of serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) were dose-dependently decreased in the drug treated animals. In addition, tea carbohydrate administration could decrease expression of vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) in H22 tumor tissue. It can be concluded that tea carbohydrate displayed strong antitumour activity in animals.     

Quantification of phototrophic biomass on rocks: optimization of chlorophyll-a extraction by response surface methodology

Biological colonization of rock surfaces constitutes an important problem for maintenance of buildings and monuments. In this work, we aim to establish an efficient extraction protocol for chlorophyll-a specific for rock materials, as this is one of the most commonly used biomarkers for quantifying phototrophic biomass. For this purpose, rock samples were cut into blocks, and three different mechanical treatments were tested, prior to extraction in dimethyl sulfoxide (DMSO). To evaluate the influence of the experimental factors (1) extractant-to-sample ratio, (2) temperature, and (3) time of incubation, on chlorophyll-a recovery (response variable), incomplete factorial designs of experiments were followed. Temperature of incubation was the most relevant variable for chlorophyll-a extraction. The experimental data obtained were analyzed following a response surface methodology, which allowed the development of empirical models describing the interrelationship between the considered response and experimental variables. The optimal extraction conditions for chlorophyll-a were estimated, and the expected yields were calculated. Based on these results, we propose a method involving application of ultrasound directly to intact sample, followed by incubation in 0.43 ml DMSO/cm² sample at 63°C for 40 min. Confirmation experiments were performed at the predicted optimal conditions, allowing chlorophyll-a recovery of 84.4 ± 11.6% (90% was expected), which implies a substantial improvement with respect to the expected recovery using previous methods (68%). This method will enable detection of small amounts of photosynthetic microorganisms and quantification of the extent of biocolonization of stone surfaces.     

黄酒中挥发性和半挥发性成分检测固相微萃取条件的优化

采用固相微萃取-气相色谱联用质谱法分析黄酒中的挥发性和半挥发性成分,对萃取时间、萃取温度、预热时间、酒样添加量、盐添加量、解析时间等条件参数进行了优化,最终确定最佳条件为采用50/30 μm DVB/CAR/PDMS萃取头,在20 mL顶空瓶中,加入6 mL黄酒样和2.0 g NaCl,萃取温度60℃,预热时间15 min,萃取时间30 min,解析温度250℃,解析时间6 min。本方法简单快速,干扰少,灵敏度高。     

响应面法优化微波辅助提取枇杷叶中黄酮类化合物

为充分利用枇杷叶,研究微波辅助乙醇浸提法从枇杷叶中提取黄酮类化合物的工艺。首先研究了影响提取效果的因素:不同体积分数的乙醇溶液、料液比、时间及微波功率。在单因素试验基础上利用响应面分析法,确定了各因素与总黄酮得率之间的关系,并以总黄酮得率为响应值做响应面和等高线图。研究表明,枇杷叶中黄酮类化合物提取最佳工艺为乙醇体积分数50%,料液比1∶30,提取时间3 min,微波功率500 W,最佳条件下总黄酮得率为2.04%,与理论推导值2.07%相比,误差为1.4%,黄酮类化合物响应方程的回归分析表明此方法合理可行。本研究对枇杷叶总黄酮提取优化的研究为进一步探讨枇杷叶的深度开发提供了理论参考依据,并为枇杷叶总黄酮的生物活性研究提供材料。     

Optimization of supercritical carbon dioxide extraction of silkworm pupal oil applying the response surface methodology

Supercritical carbon dioxide extraction (SC-CO₂) of oil from desilked silkworm pupae was performed. Response surface methodology (RSM) was applied to optimize the parameters of SC-CO₂ extraction. The effects of independent variables, including pressure, temperature, CO₂ flow rate, and extraction time, on the yield of oil were investigated. The statistical analysis showed that the pressure, extraction time, and the quadratics of pressure, extraction time, and CO₂ flow rate, as well as the interactions between pressure and temperature, and temperature and flow rate, showed significant effects on oil yield. The optimal extraction condition for oil yield within the experimental range of the variables researched was at 324.5 bar, 39.6 °C, 131.2 min, and 19.3 L/h. At this condition, the yield of oil was predicted to be 29.73%. The obtained silkworm pupal oil contained more than 68% total unsaturated fatty acids, and alpha-linolenic acid (ALA) accounted for 27.99% in the total oil.     

发酵法生产紫杉醇的检测分析

目的:采用高效液相色谱法对发酵液中的紫杉醇进行测定。方法:将紫杉醇产生菌发酵产物经乙酸乙酯萃取得测定样品,高效液相色谱分析方法为苯基柱(250mm×4.6mm,5μm),流动相乙腈-甲醇-水(36∶4∶60),流速1mL/min,检测波长227nm,进样体积20μL,柱温室温。结果:紫杉醇与干扰物可达到基线分离,在2.2～110μg/mL范围内,紫杉醇的峰面积与浓度线性关系良好,相关系数0.9996,平均回收率为99.55%,RSD为0.60%。结论:与使用C18柱色谱条件相比,该分析方法灵敏度高,不需要复杂的样品前处理过程,仪器配置不复杂、操作方便、准确性高,可有效地检测发酵液中紫杉醇的含量。     

Determination of remifentanil in human plasma by liquid chromatography-tandem mass spectrometry utilizing micro extraction in packed syringe (MEPS) as sample preparation

Remifentanil is a synthetic short-acting opioid with a short half-life that is being used during anaesthesia of small children. In this work an LC-MS/MS method for remifentanil quantification in 20 μL volume of human plasma was developed and validated in connection with a clinical study on neonatal children. Sample preparation was performed with micro extraction in packed syringe (MEPS), which is a miniaturization of solid phase extraction. For this method a mixed phase sorbent M1 (C8, cation exchange), and a protocol for basic compound extraction was followed. Remifentanil-(13)C(6) was used as internal standard. For chromatographic separation, a C18 analytical column with gradient elution was used with mobile phase consisting of aqueous 0.1% formic acid and methanol. The total analysis time was 5.0 min and the measuring range was between 0.05 and 50 ng/mL. Precision and accuracy were with acceptance criteria of ±15%. Plasma samples were stable for 5 weeks at -20°C and for 4h at room temperature while 50% was lost after 24h. This method was successfully applied for remifentanil determination in clinical samples and results agreed with a reference method. With this method using MEPS, a low limit of quantification and much reduced sample volume was obtained as compared with previous methods.